The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels. This will help understand the RPE in normal and disease states and provide us with tools for further analysis of the RPE. To this end we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE. In the past year we have made the following progress: A) To better understand the function of RPE65 and its role in RPE/photoreceptor interactions we have made an Rpe65 knockout mouse. Rpe65-deficient (Rpe65 -/-) mice exhibit profound changes in retinal physiology and biochemistry. Rod photoreceptor outer segment discs of Rpe65 -/- mice are disorganized compared to Rpe65 +/+ and Rpe65 +/- mice. Rod function, as measured by electroretinography, is abolished in Rpe65 -/- mice although cone function remains. Rpe65 -/- mice lack rhodopsin but not opsin apoprotein. Furthermore, all-trans-retinyl esters accumulate in the RPE of Rpe65 -/- mice while 11-cis-retinyl esters are absent. Abrogation of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the phenotype. Cone pigment regeneration, however, may be dependent on a separate pathway. B) With the Rpe65-deficient phenotype described above, we have begun to analyze the effect of loss of both a functional visual cycle and rhodopsin on various aspects of retinal biochemistry. C) The RPE65 promoter is RPE-specific and the minimal length for tissue-specific in vivo promoter activity is found from between - 700 and -300 bp upstream of the start site. We have identified negative control elements upstream of this region and have determined that an NF1-like DNA-binding protein binds to the proximal promoter of this gene and we are purifying candidate proteins. We have made further transgenic animals to investigate a possible positive element and negative elements outside these regions.